In collaboration with the late Dr. Markku Linnoila, NIAAA, we constructed baculoviruses encoding the allelic variants (cys/ser 23) of the human 5HT2c receptor identified in the Finnish population as linked to reduced alcoholism to examine the influence of that amino acid change on the signaling function of the receptors. Initially we examined the G-protein activation properties of the dominant cys23 allele to define optimal conditions for meaningful comparison of the two alleles. These experiments revealed an unexpectedly significant constitutive activity of the 5HT2c receptor and established a model in which G-protein interaction determined the ligand efficacy for signaling by this receptor. In comparing the two alleles, we defined a number of differences in heterologous cell expression models, and in vitro reconstitution demonstrated that the ser23 allele displayed an intrinsically greater activity in the absence of activating ligand. We extended our work on the human 5-HT receptors to understand the apparent discrepancies among investigations determining G-protein selectivity for GPCR structures in heterologous cell expression models. To examine this we compared the apparent G-protein selectivity for two human 5-HT receptors, 5HT2c and 5HT1A, activating Gq and Gi respectively. Examined by co-expression of the receptors and G-proteins in Sf9 cells, both 5HT2c and 5HT1A appeared to activate Gi significantly more than Gq. However, when receptors and G-proteins were expressed separately and examined by in vitro reconstitution, the 5HT2c displayed the expected preference for Gq, while 5HT1A preferred Gi. These experiments reveal a significant artifact of co-expression of GPCR and G-proteins in heterologous cells as a method to examine receptor signaling. We previously examined the intramolecular mechanism(s) of class3 GPCRs, by expressing the transmembrane helix bundles of the human calcium sensing receptor (hCaR) and mGluR1a without their amino-terminal domains. The hCaR 7TM structure contains multiple, interactive ligand regulatory sites for divalent metal ion, basic amino acids and the allosteric ligand (NPS568). These allosteric sites are also functioning within the full-length structure. We have tested the importance of the second extracellular loop and all charged residues in the extracelluar sequences of the hCaR. Of particular interest, mutation of a single residue in the second extracellular loop (E767) displays a high intrinsic activity, and abrogates the steeply cooperative activation of the hCaR by calcium. Mutation of the single residue K831 of the third extracellular loop is similarly, but not so strongly activating. While amino acid substitutions show that these residues participate in ionic contacts they do not appear to form an internal salt-bridge. These studies suggest that the calcium-binding ECD interacts with the 7TM core through a contact at these residues and that the ECD may be an inhibitory constraint on the activity of the 7TM core. In a collaborative investigation with Dr. Susuan Sullivan, NIDCD, we have developed a method to examine the signaling properties of the human bitter taste receptors (T2Rs) utilizing in vitro receptor activation of G-protein. We have designed baculoviral expression vectors for all 23 candidate human T2R genes and expressed them in Sf9 cells. We have also cloned the mT2R5 as a positive control for our experiments using this same expression strategy. Plasma membrane-enriched fractions prepared from baculovirus infected Sf9 cells display high-level expression of hT2R constructs and mT2R5. Using receptor-enriched Sf9 cell membranes we have applied the identical reconstitution strategy which has succeeded for every family 1 GPCR tested. We have optimized the conditions for T2R assay using the mT2R5 and bovine retinal G-protein subunits. As these initial experiments succeeded in defining optimal conditions for ??t activation by mT2R5, we have employed these for screening membrane fractions from Sf9 cells expressing the hT2R constructs to identify potential bitter tastant ligands for the candidate receptors. These screening experiments are ongoing. To date they have identified ligands for 3 of the 12 hT2R constructs screened.